Step Wise Protocol For Preparation of Adeno-Associated Virus Luciferase Stock

 Preparation of Adeno-Associated Viruses Luciferase Stock

(Huttner et al 2003, Orlowski et al 2020)

- The day before transfection, triple flasks (total surface area 500 is each seeded with one confluent 15 cm dish of HEK293T/17 (CRL-11268, ATCC) cells and incubated overnight at 37°C and 5% CO2. 

- Once 90% confluence is reached, transfection is performed by adding the following to 20 mL of room temperature DMEM: 50 μg of cis-plasmid (firefly luciferase from pCMV-GL3), 150 μg of pDG9 or pDG2 (briefly vortex), and 250 μL of PEI-max (Polysciences) solution (pH 4.5). 

- For AAV production, 66 μg of pAV plasmid and 133 μg of pHELPER is used. The solution is vortexed for 10 s and incubated at room temperature for 15 min. The transfection mix is then added to 90 mL of pre-warmed DMEM containing 2% FBS and mixed by swirling. The medium from the triple flasks is removed and replaced with the transfection mixture. 

- After 3 days of growth, the cells is harvested by vigorous tapping of the flask, transferred to a sterile container, and pelleted by centrifugation. 

- The cell pellet is resuspended in 5 mL of lysis buffer (150 mM of NaCl, 50 mM of Tris hydrochloride, pH 8.5, and 2 mM of MgCl2), and freeze-thawed three times at −80°C and 37°C with brief vortexing after each thaw cycle. 

- Non-encapsidated DNA and contaminating RNA was then digested by adding 2 μL (10 IU/μL) of Pierce Universal Nuclease (cat. no.: 88700, Thermo Fisher Scientific) followed by incubation for 30 min at room temperature. 

- The crude cell lysate is centrifuged to pellet debris, and the supernatant is reserved for iodixanol (cat. no.: D1556, Sigma-Aldrich) gradient ultracentrifugation. Virus from the cell culture supernatant is precipitated by the addition of 31.3 g of ammonium sulfate per 100 mL of supernatant followed by incubation on ice for at least 30 min. 

- The precipitate is pelleted via centrifugation, resuspended in 5 mL of lysis buffer, and then combined with the cell pellet supernatant for iodixanol gradient ultracentrifugation. Samples for ultracentrifugation are prepared in 32.4 mL of polypropylene Optiseal tubes (Beckman Coulter). 

- Viral lysates are loaded on top of discontinuous iodixanol gradients composed of 4 mL of 60% iodixanol, 4 mL of 40%, 4.9 mL of 25%, and 7.3 mL of 17% (with 1 M sodium chloride). The gradients were centrifuged at 350,333 × g (avg) for 60 min at 18°C in a Beckman type 70 Ti fixed angle rotor. Fractions (1.25 mL) are collected from the bottom of the tube and kept for virus titration. 

- Peak fractions are dialyzed in lactated Ringer’s solution (Baxter International), filtered through a 0.22 μm pore filter (Merck Millipore), and stored at −80°C.

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